广东省首例人禽流感H5N1 NS、M和NP基因序列测定及分子进化分析

The sequence determination and molecular evolutionary analysis of the NS,M and NP genes from the first proved case infected with human avian influenza virus H5N1 in Guangdong province

目的对广东省首例人禽流感H5N1的3个内部基因NP、NS和M进行克隆、测序及遗传进化分析,旨在进一步了解该病毒的来源、遗传变异及其分子进化特征。方法采集该例死亡患者尸检肺组织标本提取病毒RNA,用RT-PCR二步法进行反转录和基因扩增。产物经TA克隆到载体中进行测序并对结果进行排列、拼接,参照已发表的病毒序列,对基因进行系统进化分析。结果应用随机引物和自行设计的3对特异性引物扩增出NP、NS和M基因全长cDNA序列。该3个基因同其HA、NA一样均属于A型禽流感病毒,与国内2004-2006年浙江人源病毒及湖南禽源病毒处于同一进化分支,而与香港1997年分离株相距较远。在这3个基因中,NS的变异性最大。宿主特异性相关基因及PDZ结构域配体(PL)基序分析显示其均为禽源基因。结论感染该病例的H5N1与近年在中国不同省份分离的人禽流感H5N1的系统进化分析显示它们很可能存在共同的祖先,需进一步研究不同来源病毒的亲缘关系以及病毒基因的变化是如何影响病毒的生物学特性、毒力和跨宿主传播的。

Three internal genes（ NS, M and NP）were cloned, sequenced and undergone molecular evolutionary analysis in order to clarify the source, hereditary variation and molecular evolutionary property of these genes, the viral RNAs of these genes were extracted from the lung tissues of autopsy from the first proved cases of avian influenza in Guangdong province, and were undergone reverse transcription and genetic amplification with two steps RT-PCR. The amplified products were cloned into TA vector for the sequencing and the results were assembled into the complete cDNA sequences. Meanwhile,the phylogenetic analysis of these genes was performed in accordance with the published sequences of viruses. The full-length eDNA sequences of these 3 genes were amplified with the self-designed specific random primers. It revealed that these 3 genes were closely clustered with the avian influenza virus HSN1 isolated from humans in Zhejiang province and from poultry in Hunan province from 2004 to 2006 ,but they were less likely to evolve from the virus of Hongkong isolated in 1997. The NS gene of these viruses belonged to allele A and had a conserved 15-nt deletion resulting in a 5 amino acid loss at position 80-84 in NS protein. In addition,the NS1 protein was characterized with the avian PL motif（ESEV）and the 15 host-specific residues of M1 ,M2 and NP proteins also showed a A/China/Guangdong 01/2006 typing. In these 3 genes, great variations could be detected in NS gene. From these observations,it seems that the HSNt virus from this case may have the common ancestor with those viruses isolated from various provinces in China. however, more studies are required to explore the biologic properties of viruses, virulence and straddle host transmission.